White-clover biomass amendment as a biological conditioning strategy for radish cultivation in Mars global regolith simulant

Long-duration human missions to Mars will require crop-production systems that reduce dependence on transported growth media and fertilizers. Martian regolith may provide a locally available mineral substrate, but its poor agronomic properties constrain direct agricultural use. This study evaluated dried whole-plant white clover ( Trifolium repens L. ) biomass as a 5% (w/w) plant-derived amendment for improving Japanese 20-day daikon radish (Raphanus sativus L. ‘Kiss Me Hatsuka Daikon’) performance in Mars Global Simulant (MGS-1). Plants were cultivated for 21 days in a peat-based control, untreated MGS-1, clover-amended MGS-1, and previously cultivated MGS-1. The proportion of planted units with visible living tissue, survivor-only mean shoot height, edible biomass, and anthocyanin concentration were evaluated. Untreated MGS-1 showed a progressive decline in visual viability over the cultivation period. By DAP 21, final survival was 78.6% in clover-amended MGS-1, compared with 9.5% in untreated MGS-1, 26.2% in previously cultivated MGS-1, and 95.2% in the peat-based control. The longitudinal viability response differed significantly among treatments (treatment × DAP interaction, p < 0.001). The clover amendment also produced significantly greater survivor-only mean shoot height than untreated MGS-1, although biomass production remained substantially below that of the control. Despite reduced biomass production in the MGS-1 treatments, fresh-mass-normalized anthocyanin concentration followed the descriptive pattern: control < clover-amended < previously cultivated < untreated MGS-1. Among treatments with replicated anthocyanin measurements, concentration differed significantly (p = 0.031), with previously cultivated MGS-1 exceeding the control (p = 0.025). The untreated treatment showed the highest observed concentration, but only one tray produced sufficient tissue for analysis. These findings demonstrate that a 5% plant-biomass amendment altered the crop-response trajectory in MGS-1 from progressive loss of visible living plant presence to sustained plant persistence, even with substantial biomass limitation. The divergence between plant persistence, productivity, and anthocyanin response indicates that successful early conditioning of regolith-based substrates should not be evaluated by viability alone. Minimally processed plant biomass may therefore provide a low-input first stage in the progressive conversion of regolith from a mineral substrate associated with crop failure toward a biologically usable cultivation medium.